KpC genes

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					                              KpC genes                 ESBL-genotype                       AmpC gene(s)
Species       Strain                        CTX-M          SHV SNP          TEM SNP
E. coli       DH5a            n.d.          n.d.           n.d.             n.d.            n.d.
              901200230       n.d.          CTX-M Group 1 n.d.              238S            n.d.
              901200284       n.d.          n.d.           n.d.             104K + 238S     DHA
              901200288       n.d.          n.d.           n.d.             n.d.            ACT / MIR
              901200370       n.d.          CTX-M Group 1 n.d.              238S            n.d.
              100329-02       n.d.          CTX-M Group 1 n.d.              n.d.            n.d.
              100329-03       n.d.          CTX-M Group 9 n.d.              Wildtype        n.d.

Klebsiella    32199           n.d.          n.d.               Wildtype     n.d.            n.d.
pneumoniae    6391            Kpc 2         n.d.               Wildtype     n.d.            n.d.
              6737            Kpc 3         n.d.               Wildtype     n.d.            n.d.


     n.d. = not detected



     Genotyping of ESBL-, AmpC- and KPC-Genes

     The genotyping of ESBL-genes was performed using the Check-MDR CT101 microarray
     (Check-Points Health BV, Wageningen, The Netherlands) according to the manufacturer’s
     instructions. In brief, whole-cell DNAs were extracted from overnight bacterial cultures using
     QiaAmp DNA minikit (Qiagen, Hilden, Germany). Templates of the target β-lactamase DNA
     sequences were generated during a multiplex ligation detection reaction step. These
     templates are then amplified, and the products are hybridized in specific array tubes. The
     tubes are then inserted in the single-channel ATR03 array tube reader upon completion of
     the detection reaction, and images are acquired and interpreted with the software supplied
     by the manufacturer (Check-Points). This software automatically translates the Check-MDR
     CT101 microarray data into the presence or absence of a specific β-lactamase gene.

     The kpc genes were PCR amplified using the primers KPC-42.for (5’-atg gcc gct ggc tgg ctt
     tt-3’) and KPC-855.rev (5’-gag cgc agt cta gcc gca g-3’) leading to a 814-bp PCR
     amplification product. Two microliters of total DNA was subjected to multiplex PCR in a 50-μL
     reaction mixture. The mix contains 1× PCR buffer (10 mmol/L Tris–HCl [pH 8.3], 50 mmol/L
     KCl), 1.5 mmol/L of MgCl2, 0.125 mmol/L of each deoxynucleotide triphosphate, 10 μmol/L
     of each primer, and 2 U of AmpliTaq Gold Polymerase (Roche, Penzberg, Germany).
     Amplifications were carried out with the following PCR conditions: 10 min at 94°C and 36
     cycles of amplification consisting of 30 s at 94°C, 40 s at 52°C, and 50 s at 72°C, with 5 min
     at 72°C for the final extension. DNA fragments were analyzed by electrophoresis in a 2%
     agarose gel at 100 V for 1 h in 1× TAE (40 mmol/L Tris–HCl [pH 8.3], 2 mmol/L acetate, 1
     mmol/L EDTA) containing 0.05 mg/L ethidium bromide. Sequencing of the kpc genes were
     carried out with purified PCR amplification products (Qiatip)

     The nucleotide sequences of both strands of the amplified DNA fragment were determined
     using the same primers (KPC-42.for and KPC-855.rev ). Sequencing was performed at
     Eurofins MWG Operon (Ebersberg, Germany) using an ABI Prism 1.1 Big Dye sequencing
     kit and an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA). Nucleotide
     sequence analysis was performed using the Lasergene 8.0 software package (DNASTAR,
     Madison,     WI).   The     results   were     compared      against   the    GenBank
(http://www.ncbi.nlm.nih.gov/) database using the BLAST algorithms. KPC variants and their
SNPs were compared to the following table in order to identify the respective KPC type:



KPC-gene

           1 atgtcactgt atcgccgtct agttctgctg tcttgtctct catggccgct ggctggcttt

         61 tctgccaccg cgctgaccaa cctcgtcgcg gaaccattcg ctaaactcga acaggacttt

        121 ggcggctcca tcggtgtgta cgcgatggat accggctcag gcgcaactgt aagttaccgc

        181 gctgaggagc gcttcccact gtgcagctca ttcaagggct ttcttgctgc cgctgtgctg

        241 gctcgcagcc agcagcaggc cggcttgctg gacacaccca tccgttacgg caaaaatgcg

        301 ctggttccgt ggtcacccat ctcggaaaaa tatctgacaa caggcatgac ggtggcggag

        361 ctgtccgcgg ccgccgtgca atacagtgat aacgccgccg ccaatttgtt gctgaaggag

        421 ttgggcggcc cggccgggct gacggccttc atgcgctcta tcggcgatac cacgttccgt

        481 ctggaccgct gggagctgga gctgaactcc gccatcccag gcgatgcgcg cgatacctca

        541 tcgccgcgcg ccgtgacgga aagcttacaa aaactgacac tgggctctgc actggctgcg

        601 ccgcagcggc agcagtttgt tgattggcta aagggaaaca cgaccggcaa ccaccgcatc

        661 cgcgcggcgg tgccggcaga ctgggcagtc ggagacaaaa ccggaacctg cggagtgtat

        721 ggcacggcaa atgactatgc cgtcgtctgg cccactgggc gcgcacctat tgtgttggcc

        781 gtctacaccc gggcgcctaa caaggatgac aagcacagcg aggccgtcat cgccgctgcg

        841 gctagactcg cgctcgaggg attgggcgtc aacgggcagt aa




KPC variants and their SNPs
KPC variant       SNP-147        SNP-308        SNP-716        SNP-814      GenBank ID
KPC-2               G              C              T              C          AY034847
KPC-3               G              C              T              T          AF395881
KPC-4               G              G              G              C          FJ473382
KPC-5               G              G              T              C          EU400222
KPC-6               G              C              G              C          EU555534
KPC-7               A              C              T              T          EU729727
KPC-8               G              C              G              T          FJ234412
KPC-9               G              C              C              T          FJ624872
KPC-10              G              G              T              T          GQ140348

				
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posted:9/17/2012
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